Genetic Diversity of Lecanosticta acicola in Pinus Ecosystems in Northern Spain

Lecanosticta acicola is one of the most damaging species affecting Pinus radiata plantations in Spain. Favourable climatic conditions and unknown endogenous factors of the pathogen and host led to a situation of high incidence and severity of the disease in these ecosystems. With the main aim of understanding the factors intrinsic to this pathogenic species, a study of the population structure in new established plantations with respect to older plantations was implemented. The genetic diversity, population structure and the ability of the pathogen to spread was determined in Northern Spain (Basque Country), where two thirds of the total Pinus radiata plantations of Spain are located. From a total of 153 Lecanosticta acicola isolates analysed, two lineages were present; the southern lineage, which was prevalent, and the northern lineage, which was scarce. A total of 22 multilocus genotypes were detected with a balanced composition of both mating types and evidence for sexual reproduction. In addition to the changing environmental conditions enhancing disease expression, the complexity and diversity of the pathogen will make it difficult to control and to maintain the wood productive system fundamentally based on this forest species.


Introduction
Needle blights are currently the most serious fungal needle diseases affecting pine species worldwide. Among the main causal agents, Lecanosticta acicola (Thümen) H. Sydow, Dothistroma pini Hulbary and Dothistroma septosporum (G. Doroguine) M. Morelet are of particular concern due to the impact they have on Pinus ecosystems in the Basque Country, Spain [1]. The symptoms caused by these fungi are quite similar and, therefore, difficult to differentiate, especially when present on the same tree [1]. Severe defoliation is caused by these pathogens that results in significant growth loss when more than 25% of the needles are damaged [2,3]. Lecanosticta acicola is considered a regulated non-quarantine pest in the EU since 2019 according to the Commission implementing regulation (EU) 2021/2285 [4].
The disease caused by Lecanosticta acicola, brown spot needle blight (BSNB), was well known in Spanish Pinus radiata D. Don plantations for decades [5,6]. Until recently, BSNB had only minor impacts on native and exotic forest trees in the north of Spain. This disease was found mainly in valley bottoms, in plantations with high tree density and areas with high humidity. In the past seven years, abnormal climatic conditions favourable to the disease and potentially unknown endogenous factors made the usual silvicultural measures inefficient in mitigating its impact and progress. This pathogen species spread widely,

Sampling
This study focused on Pinus ecosystems located in the Basque Country, Spanish Atlantic climate region. In the Basque Country, conifers cover an area of 1504.59 km 2 (20.8%) over 7234 km 2 , of which 1094 km 2 (15.1% of the total surface) correspond to P. radiata (Figure 1). Field observations and sampling were conducted from spring to late autumn in 2018 and 2020.
Three sample collections were differentiated depending on the origin of the plant material. Sample collection 1 (named BC_1) was obtained from 118 different plantations of the Basque Country, representing the infected zones of the Pinus radiata provenance No. 6 ( Figure 1). Sample collection 2 (named AR_2) was obtained from 35 needle samples of Pinus species (P. brutia, P. elliottii, P. nigra, P. pinaster, P. pinea, P. ponderosa, P. sylvestris and P. taeda) produced in a French nursery and planted in 2011 in the arboretum AR20 located in Laukiz (Bizkaia) under the European project REINFFORCE (https://reinfforce.iefc.net/ es/arboreta/ar20/ accessed on 3 April 2023). AR20 arboretum was established in an abandoned nursery that produced and distributed reproductive material centralising the supply of P. radiata to the region under study. Furthermore, this place showed serious needle blight damage. Since this nursery received and grew local (provenance No. 6) and imported seeds (United State, Chile, New Zealand, France, etc.), it was considered to be a potential source of pathogen diversity.
of the Basque Country, representing the infected zones of the Pinus radiata provenance No. 6 ( Figure 1).
Sample collection 2 (named AR_2) was obtained from 35 needle samples of Pinus species (P. brutia, P. elliottii, P. nigra, P. pinaster, P. pinea, P. ponderosa, P. sylvestris and P. taeda) produced in a French nursery and planted in 2011 in the arboretum AR20 located in Laukiz (Bizkaia) under the European project REINFFORCE (https://reinfforce.iefc.net/es/arboreta/ar20/ accessed on 3 April 2023). AR20 arboretum was established in an abandoned nursery that produced and distributed reproductive material centralising the supply of P. radiata to the region under study. Furthermore, this place showed serious needle blight damage. Since this nursery received and grew local (provenance No. 6) and imported seeds (United State, Chile, New Zealand, France, etc.), it was considered to be a potential source of pathogen diversity.
Sample collection 3 (named AR_1) was obtained from 33 symptomatic needle samples from newly established P. radiata seedlings in this arboretum. This 2-year-old material (411 seedlings) was established in this location as pathogen trap plants, at a distance of 1.5 m to infected trees from AR_2, to determine the capacity of natural inoculation.
Needle samples with visible symptoms of BSNB were collected randomly from infected trees at the three sample sites (one sample consist of samples from eight to ten trees from each sampled location) and transported in a cooler box to the laboratory. The majority of samples were collected from P. radiata at sample site BC_1, since this was the most prevalent tree species in the studied area.

Pathogen Isolation
In order to obtain L. acicola isolates, needles were examined for typical erumpent fruiting bodies and when possible, five acervuli located on different needles were selected for isolations. Needle surfaces were sterilised by wiping them with a cotton swab soaked with 70% ethanol. Each acervulus was cut from a needle under a dissecting microscope using a scalpel and placed on a glass slide in a drop of sterile water. The presence of typical L. acicola conidia was verified under a compound microscope. The conidial suspension was plated on dothistroma selective medium (DSM) with streptomycin using an Sample collection 3 (named AR_1) was obtained from 33 symptomatic needle samples from newly established P. radiata seedlings in this arboretum. This 2-year-old material (411 seedlings) was established in this location as pathogen trap plants, at a distance of 1.5 m to infected trees from AR_2, to determine the capacity of natural inoculation.
Needle samples with visible symptoms of BSNB were collected randomly from infected trees at the three sample sites (one sample consist of samples from eight to ten trees from each sampled location) and transported in a cooler box to the laboratory. The majority of samples were collected from P. radiata at sample site BC_1, since this was the most prevalent tree species in the studied area.

Pathogen Isolation
In order to obtain L. acicola isolates, needles were examined for typical erumpent fruiting bodies and when possible, five acervuli located on different needles were selected for isolations. Needle surfaces were sterilised by wiping them with a cotton swab soaked with 70% ethanol. Each acervulus was cut from a needle under a dissecting microscope using a scalpel and placed on a glass slide in a drop of sterile water. The presence of typical L. acicola conidia was verified under a compound microscope. The conidial suspension was plated on dothistroma selective medium (DSM) with streptomycin using an inoculation loop [13,14]. After four days, germinating conidia were located microscopically on the surface of the medium and transferred to Petri dishes containing DSM to obtain pure, single hyphal cultures [15]. The plates were incubated at room temperature (21 • C). An individual germinated conidium per conidiomata was kept for further analyses. Long term preservation of each fungal isolate was conducted by placing two-week-old mycelium cubes in 10% glycerol at 4 • C and was maintained in the research institute collection (Neiker, Arkaute, Spain).

Pathogen Identification
Fungal tissue was scraped from the surface of 2-week-old cultures with a sterile scalpel blade. The mycelium was homogenised using a Qiagen Tissuelyser II with sterile metal beads (Ø 2.5 mm). DNA was extracted from 100 mg of lysed fungal tissue with the Plant DNA Mini Kit (Analytik Jena AG, Jena, Germany), following the manufacturer's instructions.
The integrity of the DNA in terms of quality and quantity was verified using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). DNA working stock solutions of 20 ng/µL were made for polymerase chain reaction (PCR) amplifications. All DNA was stored at −20 • C until further use.
The identity of each isolate was confirmed by species-specific conventional PCR targeting the elongation factor region [16]. The identification of the isolates was further supported by PCR amplification and sequencing of the internal transcribed spacer (ITS) region, and the translation elongation factor 1-α (TEF1) using the primers ITS1 and ITS4 [17], and EF1-728F [18] and EF1-986R, respectively, as described in van der Nest et al. [19]. PCR reactions for each region contained 20 ng DNA, 2.5 µL 10× PCR reaction buffer, 2.5 mM MgCl 2 , 400 nM of each primer, 200 µM of each dNTP and 1 U IBIAN-Taq DNA polymerase (IBIAN Technologies, Zaragoza, Spain). The reaction conditions included an initial denaturation step at 94 • C for 10 min, 35 cycles at 94 • C for 30 s, a 45 s annealing step at 56 • C for the ITS region, 52 • C for the TEF1 region, 72 • C for 60 s and a final 10 min extension at 72 • C [11] PCR products were purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany) and sequenced by Eurofins (Genomics, Konstanz, Germany). Sequencing data were edited using Finch TV software version 1.4.0 (https://finchtv.software.informer.com/1.4/, accessed on 17 January 2023) and aligned with MEGA X software version 10.0.4 (https://www.megasoftware.net/, accessed on 17 January 2023). BLAST searches for the fungal taxa were conducted on the NCBI database (National Center for Biotechnology Information NCBI, Bethesda, MD, USA) and the consensus sequences deposited in GenBank. ITS and TEF1 haplotypes were determined with TCS 1.21 software [20] and nucleotide diversity (Pi) within the Basque Country population was calculated using DnaSP v6 [21].
Phylogenetic trees were inferred using maximum parsimony (MP) and maximum likelihood (ML) analysis in MEGA X. Alignment gaps were set as additional characters with equal value and confidence levels were calculated from 1000 bootstrap replicates. The MP tree was obtained using the Tree-Bisection-Reconnection (TBR) heuristic search option and for the construction of the ML tree, the Hasegawa-Kishino-Yano nucleotide substitution model was used.
The mating type ratio in each population was calculated. This ratio was expected to be 1:1 for randomly mating populations. A chi-square goodness of fit test for a 1:1 ratio and associated p value were estimated to evaluate departure from the null hypothesis (ratio proportion 1:1).

Simple Sequence Repeat (SSR) Loci Amplification and Data Analysis
Ten microsatellite markers MD1, MD2, MD4, MD5, MD6, MD7, MD9, MD10, MD11 and MD12 designed for L. acicola were used to amplify the respective regions in the genome [22]. The PCR reaction mixture and reaction conditions with fluorescently labelled primers were carried out as described by Janoušek et al. [11,22]. For fragment analysis, PCR products were pooled into two panels and 1 µL of these multiplexed PCR products was separated on an ABI Prism 3130 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). The mobility of the SSR products was compared to those of the internal size standard, LIZ-500(-250) and allele sizes were estimated by GeneMapper 4.0 computer software (Applied Biosystems, Foster City, CA, USA). A reference sample was run on every gel to ensure reproducibility.
For each population defined by tree origin, the total number of alleles at each SSR locus was estimated. A multilocus genotype (MLG) was constructed for each isolate by combining data for each of the 10 SSR alleles obtained. The expected multilocus genotype (eMLG) was calculated based on rarefaction using the R package poppr V.2.3.0 [23,24]. Genotypic diversity was conducted for the non-clone-corrected dataset and clone-corrected dataset, in this last case with only one isolate of each MLG considered. Shannon-Wiener index of MLG diversity (H) [25], Stoddart and Taylor's diversity index (G) [26] and evenness index E5 [27] were calculated using the same R package.
The standardised index of association (rbarD) as an estimate of linkage disequilibrium was calculated to investigate the mode of reproduction [24,28]. The expectation of rbarD for a randomly mating population was zero, and significant deviation from this value would suggest clonal reproduction. Significance was tested based on 1000 permutations and conducted in the R package poppr using the clone-corrected data [24].
The standardised measure of genetic differentiation, G'st, described by Hedrick [29] was calculated to estimate subdivision among populations. This index ranged from 0 to 1, independent of the extent of population genetic variation and locus mutation rates [29]. Pairwise GST values within the clone-corrected data were calculated using the R packages strata G V.1.0.5 [30] and mmod V.1.3.3 [31].
Hedrick's standardised GST was estimated to assess population structure among these populations [29]. Statistical significance was calculated based on 1000 permutations. Hierarchical analysis of molecular variance (AMOVA) was performed to evaluate the extent of population differentiation and structure among populations, hosts species groups, and within these groups [32].
Discriminant analysis of principal components (DAPC) was performed to infer clusters of populations without considering previous tree origin criteria [33]. DAPC was conducted with the R package adegenet V. 2.0.1 [34] using the Bayesian information criterion (BIC) to infer the optimal number of groups. Important advantages of DAPC are that it maximises variation between the groups, minimises the within-group genetic variability and does not require assumptions regarding evolutionary models [33].
To assess the relationships among MLGs, minimum spanning networks (MSNs) were constructed. Bruvos's genetic distance matrix and MSNs were generated using the R package poppr V.2.3.0 [23,24]. The genetic distance described by Bruvo et al. [35] takes the SSR repeat number into account, with a distance of 0.1 equivalent to one mutational step (one repeat).

Isolation and Population Description
A total of 153 isolates were obtained. Taking into account the initial sampling strategy, 97 isolates were obtained from sampling site 1 (BC_1), 28 isolates were obtained from six Pinus species from the arboretum AR20 (AR_2) and 28 isolates were obtained from sampling site 3 (AR_1) from seedlings of P. radiata planted in late spring of 2020 in this arboretum. These seedlings were obtained from a biosafety P2 greenhouse and the absence of the disease was confirmed by morphological and molecular methods before their establishment in the arboretum.

Pathogen Identification
All 153 isolates were confirmed as L. acicola by species-specific conventional PCR targeting the elongation factor region. When analysing the ITS and TEF1 sequences, only one ITS haplotype was represented by all the isolates, and it was 100% identical (420 aligned nucleotides) to L. acicola ex-type KC012999; USA; CMW45427 [36]. Three TEF1 haplotypes (442 aligned nucleotides) ( Figure 2) were distinguished in the Basque Country population with a nucleotide diversity of Pi = 0.00021. Haplotype MZ065328 and haplotype MZ065330 differed from MZ065332 in a single base pair and in two base pairs, respectively. Representative isolates per haplotype were included in the phylogenetic analyses ( Figure 2) and deposited into GenBank. These were MZ065328 (representing two isolates: DFA1c06 and DFA5d06), MZ065330 (representing two isolates: h6a25 and h16c25) and MZ065332 (representing 149 isolates). The topologies of the ML and MP phylogenies were similar (Figure 2), where isolates representing the haplotype of MZ065330 were clustered into the northern lineage of L. acicola and were identical to the ex-type KC013002 [36]. Isolates of haplotype MZ065328 and MZ065332 were clustered into the southern lineage of L. acicola. The haplotype of MZ065332 was 100% identical to KJ938451 (south USA) [11], whereas those of MZ065328 showed a distinctive single base polymorphism.

SSR Loci Data Analysis
All primer pairs amplified the SSR loci in the L. acicola Spanish population. Three loci (MD6, MD10, MD11) were monomorphic across all 153 isolates and were, therefore, removed from the analysis (Minor Allele Frequency <0.01). The Spanish population exhibited a total of 22 MLGs. A clone-correction of the dataset was implemented to remove the bias of resampled MLG in the analysis, resulting in a total of 33 representative isolates

SSR Loci Data Analysis
All primer pairs amplified the SSR loci in the L. acicola Spanish population. Three loci (MD6, MD10, MD11) were monomorphic across all 153 isolates and were, therefore, removed from the analysis (Minor Allele Frequency < 0.01). The Spanish population exhibited a total of 22 MLGs. A clone-correction of the dataset was implemented to remove the bias of resampled MLG in the analysis, resulting in a total of 33 representative isolates (Table 1).  [25]. G, Stoddart and Taylor's Index of MLG diversity [26]. E5, evenness [27,37,38], rbarD, the standardised index of association [28] and p-value based on rbarD index.
The number of MLGs identified for each sampling site was 18 MLGs for BC_1 and 8 and 7 MLGs, for AR_1 and AR_2, respectively. This difference is related to the sampling size and the number of isolates obtained (N = 97, for BC_1, and N = 28 for AR_1 and AR_2) ( Table 1). A more appropriate estimate for richness comparison is the eMLG value, which is an approximation of the number of genotypes that would be expected after correction of the unbalanced sample size based on rarefaction. Thus, genotypic richness was lower in AR_1 and AR_2 compared with BC_1 after sample size correction (Figure 3). The BC_1 population showed the highest genotypic diversity (G = 18), followed by AR_1 (G = 8) and AR_2 (G = 7) ( Table 1). Shannon-Wiener diversity index (H) for the BC_1 population was higher (2.89) than the index for arboretum populations (AR_1 and AR_2). The BC_1 population showed the highest genotypic diversity (G = 18), followed by AR_1 (G = 8) and AR_2 (G = 7) ( Table 1). Shannon-Wiener diversity index (H) for the BC_1 population was higher (2.89) than the index for arboretum populations (AR_1 and AR_2). The values of evenness (E5) were the same for the three established populations.
The BC_1 and AR_1 populations showed significant deviation in the rbarD value from the null hypothesis of recombination, not supporting sexual reproduction (rbarD = 0.1798 and rbarD = 0.5678, respectively, with p = 0.001 in both cases). On the other hand, AR_2 showed evidence for sexual recombination (rbarD = −0.0656, p = 0.796).
An analysis of molecular variance on the clone-corrected dataset revealed no statistically significant variation among populations (p > 0.05, variation within samples p = 0.20; variation between samples p = 0.24; variation between locations p = 0.672). There was no structure in the populations. In BC_1, 7 out of the 22 MLGs identified were present in the population defined by AR location, AR_1 and AR_2, these last populations also showed, respectively, exclusive haplotypes, three in the case of AR_1 and one in the case of AR_2 (Figure 4). The discriminant analysis of principal components (DAPC) chart also showed the lack of population structure between isolates based on location ( Figure 5). respectively, exclusive haplotypes, three in the case of AR_1 and one in the case of AR_2 (Figure 4). The discriminant analysis of principal components (DAPC) chart also showed the lack of population structure between isolates based on location ( Figure 5).

Mating Identification
Mating type idiomorphs were successfully identified for 151 of the 153 isolates ( Table 2). A chi-square test of independence indicated that there was no significant difference (p ≤ 0.05) between the mating type ratios observed in the three populations. Both mating types were found in more or less equal proportions except in the AR_1 population, in which Mat 2 was more frequent (Mat-1:Mat-2 = 10:18) ( Table 2).

Discussion
In this study, an intensive sampling of pines in the Basque Country was implemented and L. acicola was exclusively detected out of the nine species described in this genus [8]. Only L. acicola was reported in Europe within the genus Lecanosticta [39] and it was the only species known to cause BSNB until 2022, when L. pharomachri was detected in plantations in Colombia causing a severe outbreak of the disease [40].
Lecanosticta acicola is currently by far the most damaging and abundant fungal pathogen present in Pinus radiata stands in the Spanish provenance region No. 6 together with Diplodia sapinea (Fr.) Fuckel 1870 [41]. The reports of L. acicola expansion in the Northern Hemisphere increased in the last 15 years, not only in a geographical dimension, but also increasing in the number of host species, and the climatic conditions in which this pathogen is detected [8,39,42]. This emerging disease escalated in incidence and severity in the last decade, affecting the sustainability of Pinus radiata ecosystems. In the Basque country, the damage caused by this pathogen accelerated a change in the forest model due to the logging of a thousand hectares a year and the mistrust concerning P. radiata sustainability under these circumstances [1].
In the studied area, three TEF1 haplotypes were identified; one clustered into L. acicola northern lineage, identical to the ex-type KC013002 [36] and two clustered into L. acicola southern lineage. The northern haplotype represented two isolates and were isolated from seven-year-old P. sylvestris and P. nigra located in Irisasi (Gipuzkoa). The two haplotypes in the southern lineages included 151 isolates, from which 149 isolates were isolated from P. radiata, P. ponderosa, P. nigra, P. sylvestris and P. brutia. Two isolates showed a unique basepair mutation at bp site 101, these were obtained from a P. radiata stand located in Amurrio (Araba). In Europe, the southern lineage of L. acicola is found in Spain and France and the northern lineage in central and northern Europe [8]. Two isolates out of 153 were part of the northern lineage; however, there are no previous records in Spain of southern and northern lineages coexisting in the same geographical area. Previously, this phenomenon had only been in France [11,39].
The predominance of the southern lineage in the studied area might be due to the northern lineage being a relatively recent introduction. The trees from which the isolates were obtained were produced in two French nurseries located in the Alps of Upper Provence (France) during 2011 and 2012, and in a nursery located in Guémené (France) in 2013, and planted in one of the arboreta established under the European project REINF-FORCE. Despite the fact that the material was subjected to phytosanitary controls prior to introduction, it is possible that the pathogen was not detected. Predominance of the southern lineage might also be related to differences in life history traits. For example, southern isolates were reported more virulent to Pinus spp. than northern ones with the exception of P. sylvestris [43]. Spore germination capacity at 32 • C for the southern isolates was successful, whereas it failed for the northern strains [44] and these differences might contribute to a lack of adaptation to higher temperatures.
Genetic diversity in the BC_1 population obtained from different plantations of the Basque Country was higher compared with the diversity in the arboretum AR20 (location of population AR_1 and AR_2). This arboretum was established in a nursery that centralised the supply of P. radiata seedlings of the region under study. This nursery was created to produce and distribute reproductive material to the forest sector. Furthermore, this place showed serious needle blight damage. Since this nursery received and grew local and imported seeds in the past, it was considered to be a potential source of pathogen diversity and dispersal through the seedlings to the entire region. Anthropogenic movement of infected plant material and seedlings is considered the main source of long-distance dispersal of L. acicola [8]. The 28 isolates from population AR_2 with eight haplotypes from pine species established twelve years ago, and AR_1 with 7 haplotypes obtained from newly established seedlings in the arboretum, support the hypothesis of their high and fast colonisation capacity of different hosts but mainly P. radiata, which shows a high susceptibility to disease in the region [1].
Indication of sexual recombination in the sampled region was supported by the fact that both mating types were identified in more or less equal proportions in the populations of L. acicola, the high levels of observed genetic diversity, and by many of the isolates with the same multilocus haplotypes having different mating types in the same populations. Direct evidence for the sexual state of the pathogen already exists in a location 0.53 km far away from the arboretum [12] and it may also be present in these areas. Population structure analysis showed no evidence of population subdivision. It is likely that there is only one panmictic population present throughout all locations. However, a more intensive sampling of these areas may reveal new hypotheses about population structure, as was observed in other fungal species in the region [41].
The seeds and plants used in Northern Spain come from distributors in the United States, France, Denmark, New Zealand, Chile, etc., which makes it difficult to generate hypotheses about the potential origin of the pathogen's introduction [45]. Nevertheless, considering that the main pine species in the studied region is Pinus radiata and that the pathogen is absent in Chile and New Zealand, either the United States and/or France could be the main candidates for the pathogen introduction into Spain. Previous population analysis established that the origin of the northern and southern lineages present in Europe were from North America [11,46,47]. The Basque Country was the first location in Europe where the presence of L. acicola was confirmed, and where North America was potentially considered the source of the infected host plants [39]. The presence of the isolates from the northern lineage in our area could be a relatively newer introduction from other European countries caused by the northern lineage spreading within Europe through separate introductions, and thus defining characteristic populations [46,47].
The knowledge of the origin, diversity and genetic structure of pathogen populations at a global and local scale can have a remarkable impact on landscape-level planning models and other decision support systems that enable forest managers to generate optimal disease management strategies. The high levels of genetic diversity of the pathogens would complicate the implementation of successful control measures and breeding programs, and could enhance the capacity of adaptation of the pathogen to stressful conditions. In this context, preventative methods should be directed to reduce the movement of plants among countries and regions to avoid the introduction of new genetic sources of diversity into existing populations. This is even more important now in Spain, seeing that a possible recent introduction of a northern lineage was discovered in this study and that adaptation of isolates in each lineage to local climatic conditions could contribute to the success of the pathogen [8]. Some areas are so devastated by the disease that restrictions in the use of highly susceptible pine species might need to be restricted in plantations to help reduce inoculum pressure and the species becoming reservoirs of the pathogen.